The embodiment of the present invention entails a novel combination of separately existing chemistries and instrumentations to offer an ultrasensitive method for the quantitation of an analyte of interest such as a protein immobilized on a particulate solid phase, i.e., the surface of a microparticle, which serves as a carrier, support, or matrix. Due to amine-reactive nature of the signal label, any analyte of interest other than protein and having at least one amine group can be used in this process. As used hereinafter the term protein refers not only to a protein, but also to any substance of interest having at least one amine group. An amine can be selected from the group consisting of aliphatic amines, aromatic amines, diamines, polyamines, and substituted amines, e.g., acetamidyl, amidyl, or aminyl.
It is a further object of this invention to provide means of detecting and measuring such analytes. Analytes readily comprise most diverse classes of chemicals including proteins, peptides, prosthetic proteins, biogenic amines, various drugs with amine residues, transaminated nucleic acids, etc. Analytes also comprise fluorescent dyes having an amine group(s), e.g., phycobiliproteins, bound to a microparticle and is either labeled with another amine-reactive dye such as fluorescein isothiocyanate (FITC) or not labeled. One skilled in the art easily recognizes other similar substances like peptide nucleic acids having nucleobases attached to a polyamide backbone and containing alkylamine side chains as disclosed for example in the U.S. Pat. No. 5,786,461 and incorporated herein by way of reference.
The preferred method of making a standard microparticle preparation with a known average amount of a reference substance bound thereto comprises the steps of binding or associating a known average amount of a reference substance to a microparticle; and measuring the amount of microparticle-associated or bound reference substance by a standard calorimetric protein assay.
The preferred steps involved in this process are preferably, but not necessarily, carried out in the following sequence: immobilizing an analyte of interest on a particulate solid phase (e.g., microparticle) either by adsorption or by covalent conjugation; covalently binding to the analyte of interest a light-emitting signal molecule or signal label; comparing the intensity of the light emitted from the label to standard microparticle preparations with known amount of reference material or substance, which is labeled with the same light-emitting label under essentially same conditions, and determining the presence and/or amount of the analyte of interest. Typical standards will contain varying amounts of an immobilized reference protein extending across attogram to femtogram concentrations. For example, a series of standards ranging from 0.1 to 1000 femtograms per unit solid phase are sufficient for most common practical applications
More specifically a process is provided for determining the relative average amount of at least one protein of interest, which is immobilized on each of a plurality of particulate solid phases, comprising: subjecting under substantially the same protein labeling conditions a plurality of particulate solid phases to each of which is immobilized an unknown.average amount of at least one protein of interest and a plurality of standard particulate solid phases to each of which is immobilized a known average amount of at least one reference protein, to provide protein of interest and reference protein both labeled with at least one light-emitting label; and comparing the average amount of light emitted by labels found on the standard particulate solid phases harboring the reference protein with that emitted by labels found on particulate solid phases harboring the protein of interest, to provide a relative average amount of the protein of interest immobilized on each of the particulate solid phases.
An alternative preferred method of measuring the concentration of an analyte bound to a particulate solid phase is also provided. This method consists of associating the analyte of interest with the particulate solid phase, e.g., microparticle, which has a light-emitting label embedded within or immobilized thereon. The label is chosen in such a way that it is capable of changing its light absorption and/or emission pattern as a function of the concentration of the analyte. In addition at least one and preferably more reference samples are prepared with the known amount of the reference substance bound to the solid phase. This solid phase has the same light-emitting label and the reference substance is bound to the microparticle by essentially identical procedure as the analyte. The measurement of the amount of bound analyte is then achieved by comparing the light emission characteristics or the optical signal with the light emission spectra (optical signal) of the reference sample(s).
The preferred method comprises quantifying the amount of at least one protein of interest immobilized directly or indirectly on a particulate solid phase comprising, subjecting one or more particulate solid phases, to each of which is immobilized directly or indirectly an unknown amount of at least one protein of interest, to labeling conditions effective to affix to each particulate solid phase an effective amount of a light-emitting label to provide a labeled amount of the at least one protein of interest which is proportional [sufficient to label] to the amount of the at least one protein of interest immobilized to the particulate solid phase; and elating the amount of light emitted from each particulate solid phase to the amount of the at least one protein of interest immobilized to the particulate solid phase using a standard curve. The average amount of the reference substance bound to the microparticle is in a range between about 0.01 and 1,000 femtograms per microparticle.
The preferred method of measuring the quantity of an analyte bound to a particulate solid phase, said method comprising the steps of binding an analyte with the particulate solid phase, said particulate solid phase having a fluorescent label capable of emitting a fluorescent signal embedded within said particulate solid or immobilized thereon, said label being capable of changing its fluorescence signal as a function of the concentration of the analyte; providing at least one standard microparticle preparation with a known average amount of a reference substance bound thereto, said standard microparticle preparation having the same fluorescent label as said particulate solid phase and measuring the quantity of the analyte [associated with] bound to the particulate solid phase by comparing the fluorescence signal emitted from said particulate solid phase with the fluorescence signal emitted from the standard microparticle preparation.
To achieve higher precision it is preferable that the reference substance is labeled with the light-emitting label under the same or essentially the same reaction condition as the analyte. Typically, this means that both analyte (protein) and reference substance (protein) are labeled at the same time and in the same reaction mixture. To achieve even better precision it is preferable that both analyte and reference substance are same or closely related classes of proteins. It is preferable that reference protein is the same as, similar to, analogous to, homologous to, or functionally equivalent to the protein of interest. For example if the analyte is an immunoglobulin it is preferable that the reference substance is also an immunoglobulin of the same isotype. If a single standard is available only at one known concentration then one can only make inferences about the relative amount of the analyte (protein) of interest. Better precision and absolute quantitation is possible when a plurality of standards with known amounts of reference proteins are available which permits constructing a standard curve and based on that one skilled in the art can interpolate the quantity of the analyte of interest.
The invention, based on measuring precisely the number or concentration of analyte molecules on a microparticle is useful for a wide variety of applications including but not limited to manufacturing of precision-coated microspheres, quality control, immunoassays, diagnostic procedures, therapeutic uses, affinity purification, environmental applications, recombinant DNA technology, drug and enzyme applications. The particulate solid phase based reagent is intended for use in assay systems such as radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescent immunoassay (FIA), luminescence immunoassay (LIA), calorimetric immunoassay, cytoimmunochemistry, cell sorting, agglutination, and alike. Other applications requiring the knowledge of the exact number or concentration of analyte molecules can be imagined and are readily recognized by those skilled in the art. As used hereinafter the term concentration refers interchangeably to the number, quantity, or amount of molecules of interest.
A method adapted to flow cytometry procedure is provided as a specific example although one skilled in the art will readily recognize that other technically equivalent procedures of measuring an analyte of interest are adaptable to exploit the disclosed invention. These include devices capable of measuring an emission light and selected from the group consisting of a microscope equipped with light-measuring device, a fluorometer, a spectrophotometer, a luminescence, or scintillation counter. Other equivalent means comprise visual inspection, digital (CCD) cameras, video cameras, photographic film, or the use of current instrumentation such as laser scanning devices, photodiodes, quantum counters, plate readers, epifluorescence microscopes, scanning microscopes, confocal microscopes, capillary electrophoresis detectors, a photomultiplier tube or light detector capable of detecting the presence, location, intensity, excitation and emission spectra, fluorescence polarization, fluorescence lifetime, and other physical properties of the light-emitting signal. These physically distinguishable optical features in combination with size and shape of the particle will characterize each distinct set of microparticles. Other properties such as magnetic inclusions in the body of the particle can be imagined by one skilled in the art.
Also provided is the method of preparing standard microparticle preparation carrying the known amount of immobilized protein or any other suitable reference substance, which is labeled in a manner and under conditions similar or identical to the processing of the analyte. Although the method combines known in the art procedures the final product, which is a microparticle with precisely defined amount of reference substance is uniquely distinct from any existing microparticle in the prior art and thus it is also an object of the invention. In the past the precise measurement of the reference substance was difficult to achieve, especially in ranges that are below sensitivity thresholds of prior art methods. The amount of bound reference substance can be as low as one attogram. For practical purposes the lower level can start in femtogram range and extend into microgram or higher ranges.
It should be apparent that another object of this invention is to provide a set of standard microparticles with known amounts of immobilized reference substance which would be incorporated in a kit for analyte assay. The microparticles themselves may contain additional, one or more fluorophores embedded at or inside the solid phase to help to distinguish one set of particles from another.
In accordance with the subject invention the preferred embodiment involves the solid phase immobilized protein standards useful for the invention and exhibit one or more, preferably all, of the following characteristics. The solid phase (microparticle) is highly uniform and suited for analysis in a flow analyzer (e.g., a suitably equipped flow cytometer), usually 1-20 xcexcm in diameter, preferably 3-9 xcexcm; has a standard reference protein associated with it; and has immobilized protein concentrations between about 0.01 and about 1000 femtograms or higher. The lower limit can be even lower and can easily extend into attogram range. The solid phase is preferably stable with respect to size and surface protein concentration in suspending media (however, for particular applications, such as sustained release particles designed for drug delivery, the stability is not critical, because the drug is intended to be gradually released). In addition; the solid phase may contain additional fluorophores or light-emitting dyes at or within the solid phase to correspond to specific subsets of the set.
An essential embodiment of the invention is a particulate solid phase to which is bound a known average amount of at least one reference protein, the known average amount ranging in value from about 10xe2x88x929 to about 10xe2x88x9218 grams. More preferably the known average amount ranges in value from about 10xe2x88x9212 to about 10xe2x88x9218 grams. Even more preferably the known average amount ranges in value from about 10xe2x88x9214 to about 10xe2x88x9218 grams. The embodiment further comprises an average amount of at least one light-emitting label, which average amount is proportional to the known average amount of the at least one reference protein and is further characterized by one or more distinguishing properties that permit the classification of the particulate solid phase as belonging to a particular set having a predetermined known range or known average amount of the at least one reference protein. In addition it is understood that one or more distinguishing properties are defined by the size of the particulate solid phase, its shape, its color, magnetic properties, optical emission characteristics, or combinations thereof. Preferably the particulate solid phase emits light at one or more predetermined wavelengths or within predetermined ranges thereof
In a preferred embodiment the microparticle immobilized analyte assay is carried out by using a kit. Such a kit is useful for determining the concentration of a protein immobilized on a particulate solid phase comprising first container containing a light-emitting label having at least one reactive functional group; and one or more second containers each containing a plurality of standard particulate solid phases harboring a known average amount of at least one reference protein. Such a kit can be minimal containing only essential proprietary component not available commercially from other sources, i.e., standards preparation. This preparation will be then processed by a practitioner in parallel with the unknown amount of analyte of interest according to the process as disclosed in detail infra. Alternatively, such a kit is essentially a complete kit containing all necessary components in one package and consisting of various buffers and reagents, light-emitting label stock, uncoated microparticles, and microparticle immobilized standards or reference substance.
These and other objects of the invention will become apparent to those of ordinary skill in the art, especially after consideration of the following detailed description of the preferred embodiments.